The control of cell proliferation and survival are critical factors in maintaining healthy cell populations and eliminating those cells defective in proper cell cycle control. A key protein regulating the cell cycle and apoptosis is p53, a protein mutated in >50% of human cancers. We recently found that the combined loss of both p53 and stathmin, a microtubule destabilizing protein, results in a cell cycle delay during the G2 phase of the cell cycle and cell death by apoptosis. These data provide insight into controls governing cell proliferation and hold the potential to identify targets to kill specifically those tumor cells lacking functional p53. Experiments proposed in this application look to identify the signal pathway linking reduced stathmin level and delayed progression through the G2 phase of the cell cycle. Completion of the proposed aims will test the hypothesis that stathmin depletion stabilizes the interphase microtubule cytoskeleton, which acts downstream to limit Rho-dependent activation of Aurora A and mitotic entry. Throughout the aims, alternative models are also tested. In Aim 1, the signal relay downstream of stathmin depletion will be identified. Drug-induced microtubule depolymerization or stabilization will be used to test for their ability to abrogate (depolymerization) or mimic (stabilization) the observed delay in G2. The other stathmin binding partners, p27Kip1 and STAT3, will be tested for their potential role(s) in regulating cell cycle progression after stathmin depletion. Over-expression of stathmin truncations will be used to further probe for microtubule- dependent or -independent regulation of interphase cell cycle progression. In Aim 2, pathways controlling mitotic entry will be examined for their role in relaying signals after stathmin depletion. The levels of cyclins and their rate of accumulation will be measured, the activity states of enzymes directly controlling mitotic entry will be examined, and finally, upstream signal cascades will be manipulated to either abrogate or mimic the stathmin depletion-induced delay in G2. Completion of the two aims will identify the signal cascade linking stathmin depletion and mitotic entry. PUBLIC HEALTH RELEVANCE: Controlling cancer cell proliferation requires interventions that can block cell replication, ideally without impacting the non-transformed cells in the body. We have identified the microtubule destabilizing protein, stathmin, as a protein necessary for reproduction and survival of cancer cells lacking the tumor suppressor p53. Since about half of all human cancers harbor mutations in p53, understanding why stathmin is required for cell proliferation in these cells holds great potential for selectively controlling their growth.